Mass spectrometry has become the method of choice for fast and efficient identification of proteins in biological samples. Tandem mass spectrometry of peptides in a complex protein mixture can be used to identify and quantify the proteins present in the original mixture. Tandem mass spectrometers achieve this by selecting single m/z values and subjecting the precursor ions to fragmentation, providing product ions that can be used to sequence and identify peptides. The information created by the product ions of a peptide can be used to search peptide and nucleotide sequence databases to identify the amino acid sequence represented by the spectrum and thus identify the protein from which the peptide was derived. Analytical methods that compare the fragment ion pattern to theoretical fragment ion patterns generated computationally from sequence databases can be used to identify the peptide sequence. Such methods can identify the best match peptides and statistically determine which peptide sequence is more likely to be correct. The algorithms typically utilize mass-to-charge ratio (m/z) information for identification purposes of the various product ions.
Fragmentation can be provided by various methodologies and mechanisms. Ion activation techniques that involve excitation of protonated or multiply protonated peptides, include collision-induced dissociation (OD), and infrared multiphoton dissociation (IRMPD) for example, and have been used to identify sequences. In these dissociation methods, translational energy is imparted to the peptide and is converted into vibrational energy that is then distributed throughout the bonds of the peptide. When the energy imparted to a particular bond exceeds that required to break the bond, fragmentation occurs and product ions are formed. The cleavage may not always however, occur along the backbone of the peptide if, for example, the side-chain of the peptide has elements that inhibit cleavage along the backbone, by providing a lower energy pathway and cleavage site on a side-chain. This preferential cleavage of the side-chain bonds rather than the polypeptide bonds often results in the provision of information primarily about the side-chain sequences and not the peptide sequence.
Other mechanisms of fragmentation include for example, those in which the capture of a thermal electron is exothermic and causes the peptide backbone to fragment by a non-ergodic process, those that do not involve intramolecular vibrational energy redistribution. Such methodologies include Electron Capture Dissociation (ECD) and Electron Transfer Dissociation (ETD). ECD and ETD occur on a time scale that is short compared with the internal energy distribution that occurs in the CID process, and consequently, most sequence specific fragment forming bond dissociations are typically randomly along the peptide backbone, and not of the side-chains. Ions from side chain cleavage are generally not observed and thus ECD and ETD are thought to provide more complete information on primary structure of peptides. However, in addition to the fragment ions, peaks are generally seen for ions which have been subjected to neutral loss, such as water (−18 Da) for example.
Unfortunately, ECD cannot be performed with trap-type mass analyzers since the electrons created by the reaction do not typically retain their thermal energy long enough to be trapped, thus ECD is typically performed on an FT-ICR mass spectrometer. These instruments are expensive. ETD fragmentation provides an alternative to ECD that can be performed on the more-readily-available ion trap instruments. In ETD, analyte ions are reacted under controlled conditions with reagent ions of opposite polarity. The transfer of electrons between reagent and analyte ions (from the reagent ion to the analyte ions for analyte cations) produces dissociation of the analyte ions.
FIG. 1 depicts a nomenclature typically adopted (and used herein) for the fragments of peptides and proteins (see Roepstorff and Fohlman [Roepstorff, 1984], and Johnson et. al. [Johnson, 1987], both of which are incorporated by reference herein). The three possible cleavage points of the peptide backbone are called a, b and c when the charge is retained at the N-terminal fragment of the peptide and x, y and z when the charge is retained by the C-terminal fragment. The numbering indicates, which peptide bond is cleaved counting from the N- and the C-terminus respectively, and thus also the number of amino acid residues in the fragment ion. The number of hydrogens transferred to or lost from the fragment is indicated with apostrophes to the right and the left of the letter respectively. Peaks corresponding to ions that have lost ammonia (−17 Da) and water (−18 Da) are denoted, respectively, by superscript asterisks and circles to the right of the letter.
It has been observed that low-energy CID predominantly yields, when fragmentation is along the peptide backbone, fragment ions (ion products) of type a, b, and y; a*, b*, and y* and a°, b°, and y°. By contrast, ETD produces mainly c and z* fragment ions and to a much smaller extent a*, y ions and z′ and c* ions. Thus, the two techniques can yield complementary information when performed on the same precursor ions during the same scan event. However, such mixed-fragment mass spectra can be difficult to model and interpret, using conventional analysis techniques. This is mainly because the higher number of features present in the spectra would have a higher chance of matching with decoy spectra. This would cause an increase in the false discovery rate. Conventional analysis techniques are designed to deal with spectra containing only one type of fragments (c/z or b/y). Thus, such mixed-fragment mass spectra require a different search approach than is generally employed in analyses of conventional spectra, in which only one type of fragments is expected.